Stimulation of an immune response by enantiomers of cationic lipids

ABSTRACT

A composition and method for activating immune cells ex-vivo, or inducing an immune response in a subject including a composition comprising at least one chiral cationic lipid. The chiral cationic lipid in one embodiment comprises a nonsteroidal cationic lipid having a structure represented by formula (I); wherein in R 1  is a quaternary ammonium group, Y 1  is a space chosen from a hydrocarbon chain, an ester, a ketone, and a peptide, C* is a chiral carbon, R 2  and R 3  are independently chosen from a saturated fatty acid, an unsaturated fatty acid, an ester-linked hydrocarbon, phosphor-diesters, and combination thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/899,763 filed Jun. 12, 2020, which is a continuation U.S. patentapplication Ser. No. 15/702,063 filed Sep. 12, 2017, now issued as U.S.Pat. No. 10,702,541 on Jul. 7, 2020, which is a divisional of U.S.patent application Ser. No. 12/988,236 filed Dec. 23, 2010, now issuedas U.S. Pat. No. 9,789,129 on Oct. 17, 2017, which is a 371 ofInternational Patent Application. No. PCT/US2009/040500 filed Apr. 14,2009, which claims benefit of U.S. Provisional Application No.61/045,837 filed Apr. 17, 2008, the disclosures of which areincorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 16, 2021, isnamed PDS-16-1033WO-US-DC-CON_SL.txt and is 648 bytes in size.

FIELD OF THE INVENTION

The present invention generally related to stimulating an immuneresponse, and more particularly to the use of the R and S enantiomers oflipids in stimulating immune responses.

BACKGROUND OF THE INVENTION

This section is intended to introduce the reader to various aspects ofart that may be related to various aspects of the present invention,which are described and/or claimed below. This discussion is believed tobe helpful in providing the reader with background information tofacilitate a better understanding of various aspects of the presentinvention. Accordingly, it should be understood that these statementsare to be read in this light, and not as admissions of prior art.

Development of safe and effective immunotherapies for human use remainsan urgent medical need for patients worldwide. In order to elicitappropriate immune responses, immunologic modifiers (“immunomodifiers”)that enhance, direct or promote an immune response can be used invaccine design or immunotherapy [Gregoriadis, G., Immunologicaladjuvants: a role for liposomes, Immunol Today 11:89 (1990)]. Forexample, vaccines may include antigens to stimulate an immune response.However, some potential vaccines that include antigens are weakstimulators of an immune response because the vaccines do notefficiently deliver the antigen to antigen presenting cells (“APC”) ofthe immune system and/or the antigen is weakly immunogenic. Thus,immunotherapies that effectively deliver antigens to APC, and alsostimulate the immune system to respond to the antigen, are needed.Immunomodifiers have the potential to function as such an immunotherapy.Such immunotherapies may have these and other benefits. For example,when included as part of a therapeutic vaccine, an immunomodifier shouldat least (1) improve antigen delivery and/or processing in the APC[Wang, R. F., and Wang, H. Y. Enhancement of antitumor immunity byprolonging antigen presentation on dendritic cells. Nat Biotechnol20:149 (2002)1, (2) induce the production of immunomodulatory cytokinesthat favor the development of immune responses to the vaccine antigen,thus promoting cell mediated immunity, including cytotoxic T-Lymphocytes(“CTL”), (3) reduce the number of immunizations or the amount of antigenrequired for an effective vaccine [Vogel, F. R. Improving vaccineperformance with adjuvants. Clin Infect Dis 30 Suppl 3:S266 (2000)], (4)increase the biological or immunological half-life of the vaccineantigen, and (5) overcome immune tolerance to antigen by inhibitingimmune suppressive factors [Baecher-Allan, C., and Anderson, D. E.Immune regulation in tailor-bearing hosts. Curr Opin Immunol 18:214(2006)].

Presently, the primary class of agents used to enhance the efficacy ofantigens, such as peptide or protein antigens, in eliciting an immuneresponse are adjuvants such as water-in-oil emulsions, alum, and otherchemicals which enhance antigen responses; however, these adjuvants arenot immunomodifiers, as described above, because they have no directimmunomodulatory effects themselves [Vogel, F. R., and Powell, M. F. Acompendium of vaccine adjuvants and excipients, Pharm Biotechnol 6:141(1995)1. Several such adjuvants are available for use in animals andsome of them have been tested in clinical trials. In addition totraditional adjuvants such as the aluminum salts, products such asinfluenza virosomes [Gluck, R., and Walti, E. 2000. Biophysicalvalidation of Epaxal Berna, a hepatitis A vaccine adjuvanted withimmunopotentiating reconstituted influenza virosomes ORM. Dev Biol(Basel) 103:189 (2000)1, and Chiron's MF59 [Kahn, J. 0., et al. Clinicaland immunologic responses to human immunodeficiency virus (I ⁻1.17) typeISF2 gp120 subunit vaccine combined with MF59 adjuvant with or withoutmummy! tripeptide dipalinitoylphosphatidylethanolamine in non-I ⁻IIV-ittfected human volunteers. J Infect Dis 170:1288 (1994)], whichhave intrinsic immune effects, are being marketed. For example, MF59,which is a submicron emulsion based adjuvant, is internalized bydendritic cells [Dupuis, M., et al., Dendritic cells internalize vaccineadjuvant after intramuscular injection. Cell Immunol 186:18 (1998)].However, according to clinical trial reports on HSV and influenzavaccines [Jones, C. A., and Cunningham, A. L. Vaccination strategies toprevent genital herpes and neonatal herpes simplex virus (HSV) disease.Herpes 11:12 (2004); Minutello, M. et al., Safety and immunogenicity ofan inactivated subunit influenza virus vaccine combined with ilifF59adjuvant emulsion in elderly subjects, immunized for three consecutiveinfluenza seasons. Vaccine 17:99 (1999)1, evidence from animal modelssuggests that the MF59 adjuvant enhances production of neutralizingantibodies rather than enhancing responses. Thus, new methods ofstimulating cell mediated immune responses are needed.

Further, as mentioned above, some antigens are weak stimulators of animmune response. Thus, in addition to co-administering antigen withsubstances that stimulate immune responses, as described above, a weaklyimmunogenic antigen can be modified to increase its immunogenicity. Forexample, a weakly immunogenic antigen can be coupled to immunogenicpeptides, polysaccharides, or lipids to increase its immunogenicity.However, simply coupling weakly immunogenic antigens to these types ofcompounds may not be sufficient to elicit an immune response. Forexample, the resulting immune response may be directed to immunogenicepitopes on the coupled compound and not the weak antigen, or thecoupled antigen may not be efficiently delivered to APC of the immunesystem. Thus, additional methods are needed to stimulate immuneresponses to antigens that are weakly immunogenic.

SUMMARY OF THE INVENTION

Certain exemplary aspects of the invention are set forth below. Ttshould be understood that these aspects arc presented merely to providethe reader with a brief summary of certain forms the invention mighttake and that these aspects are not intended to limit the scope of theinvention. Indeed, the invention may encompass a variety of aspects thatmay not be explicitly set forth below.

This invention is directed to the chirality of cationic lipids and theuse of the R and S enantiomers of cationic lipids, which under certaindose and composition conditions act as a novel class ofimmune-stimulants, to (1) effectively present or deliver an antigen tothe immune system and (2) stimulate the immune system to respond to theantigen.

Liposomes have been extensively used for delivering small molecularweight drugs, plasmid DNA, oligonucleotides, proteins, and peptides.Vaccines using liposomal vehicles as nonviral antigen carriers arepreferable compared to traditional immunizations using live attenuatedvaccines or viral vectors such as vaccinia or influenza virus. U.S.patent application Ser. No. 12/049,957, assigned to the assignee of thepresent application, discloses simple yet effective lipid-basedimmunotherapies, including a cationic lipid/antigen complex, which hastwo molecules, a cationic lipid and an antigen, and the effects of thelipid dose on the resulting immune response. The reported resultsdemonstrate that the cationic liposome complexed with an antigen servesto stimulate immune responses and initiate dendritic cell (an APC)interaction with T-cells.

In the present invention, additional studies performed with the twoenantiomers of a selected cationic lipid have led to the discovery thatdifferences exist in the ability of the R and S enantiomers of thecationic lipids to act as potent immune activators under variousconditions. In combination with an antigen, the cationic lipid/antigencomplex containing the R enantiomer, under various dose conditions(including low dose conditions), induces strong immune responsesspecific to the antigen formulated in the complex and results in tumorregression. Complexes consisting of S-DOTAP and the antigen however wereable to induce only limited tumor regression, and not at all doses atwhich R-DOTAP was effective. Both enantiomers of DOTAP are howeverequally effective at inducing maturation and activation of dendriticcells, which is the first step in inducing a cellular immune response.

Thus, one aspect of the invention provides a composition of at least oneenantiomer of a cationic lipid in a dose sufficient to induce an immuneresponse in a subject.

Another aspect of the invention provides a method of inducing an immuneresponse in a subject by administering a specific enantiomer or amixture of enantiomers of a cationic lipid to the subject.

Another aspect of the invention provides a composition of an R or Senantiomer of a cationic lipid in a dose sufficient to induce an immuneresponse in a subject.

Additional aspects of the invention involve the addition of at least oneantigen to the R or S enantiomer to form a cationic lipid/antigencomplex in which case the immune response is antigen-specific.

BRIEF DESCRIPTION OF THE DRAWINGS

Various features, aspects, and advantages of the present invention willbecome better understood when the following detailed description is readwith reference to the accompanying figures, wherein:

FIGS. 1A and 1B depict chirality of I,2-dioleoyl-3-trimethylammoniumpropane (“DOTAP”).

FIG. 2 is a graph depicting activation of human dendritic cellsresulting in expression of the co-stimulatory molecule CD 80 by R-DOTAP,S-DOTAP and the racemic mixture RS-DOTAP.

FIG. 3 is a graph depicting activation of human dendritic cellsresulting in expression of the co-stimulatory molecule CD 83 by R-DOTAP,S-DOTAP and the racernic mixture RS-DOTAP.

FIG. 4 is a graph depicting activation of human dendritic cellsresulting in expression of the co-stimulatory molecule CD 86 by R-DOTAP,S-DOTAP and the racemic mixture RS-DOTAP.

FIG. 5 is a graph depicting stimulation of human dendritic cellsresulting in production of the chemokine CCL-3 by R-DOTAP, S-DOTAP andthe racemic mixture RS-DOTAP.

FIG. 6 is a graph depicting stimulation of human dendritic cellsresulting in production of the chemokine CCL-4 by R-DOTAP, S-DOTAP andthe racemic mixture RS-DOTAP.

FIG. 7 is a graph depicting stimulation of human dendritic cellsresulting in production of the chemokine CCL-5 by R-DOTAP, S-DOTAP andthe racemic mixture RS-DOTAP.

FIG. 8 is a graph depicting stimulation of human dendritic cellsresulting in production of the chemokine CCL-19 by R-DOTAP, S-DOTAP andthe racemic mixture RS-DOTAP.

FIG. 9 is a graph depicting stimulation of human dendritic cellsresulting in production of the cytokine IL-2 by R-DOTAP, S-DOTAP and theracemic mixture RS-DOTAP.

FIG. 10 is a graph depicting stimulation of human dendritic cellsresulting in production of the cytokine IL-S by R-DOTAP, S-DOTAP and theracemic mixture RS-DOTAP.

FIG. 11 is a graph depicting stimulation of human dendritic cellsresulting in production of the cytokine 1L-12 by R-DOTAP, S-DOTAP andthe racemic mixture RS-DOTAP.

FIG. 12 is a graph demonstrating the in vivo antitumor effect of variousdoses of a cationic lipid/antigen complex based on tumor size and. timepost-injection.

FIG. 13 is a graph demonstrating the effect of S-DOTAP dose on the invivo anti tumor efficacy of the cationic lipid/antigen complex.

FIG. 14 is a graph demonstrating the effect of R-DOTAP dose on the invivo anti tumor efficacy of the cationic lipid/antigen complex.

FIG. 15 is a graph depicting the lipid dose response effects of theracemic mixture of DOTAP, R-DOTAP and S-DOTAP on the in vivo anti-tumorimmune response of the cationic lipid/antigen complex with antigen doseof 201.1 g. The effect of antigen dose is also demonstrated with theracemic mixture of DOTAP. R-DOTAP compared to S-DOTAP: * p<0.05, **p<0.01, n=5-6.

DETAILED DESCRIPTION

One or more specific embodiments of the present invention will bedescribed below. In an effort to provide a concise description of theseembodiments, all features of an actual implementation may not bedescribed in the specification. It should be appreciated that in thedevelopment of any such actual implementation numerousimplementation-specific decisions must be made to achieve thedevelopers' specific goals, which may vary from one implementation toanother. Moreover, it. should be appreciated that such a developmenteffort might be complex and time consuming, but would nevertheless be aroutine undertaking for those of ordinary skill having the benefit ofthis disclosure.

When introducing elements of the present invention (e.g., the exemplaryembodiments(s) thereof), the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

One aspect of the present invention provides an enantiomer of a cationiclipid to stimulate an immune response in a mammal to prevent or treatdisease The individual chiral lipids can function independently asimmunomodulators, in a dose dependent manner, such as for production ofchemokines and/or cytokines, by activating various components of the MAPkinase signaling pathway. The dose range that effectively induces animmune response is observed to differ between the R and S enantiomersand also within various mammalian species. For example, in the rodentspecies the R-enantiomer of DOTAP effectively attenuates tumor growthover a range of about 30 nmole to about 400 nmole. In contrast, theS-enantiomer of DOTAP is effective over this same range of doses in thesame species of rodent, though less so than the R-enantiomer. In anotheraspect, the chiral cationic lipid may be associated with antigens ordrugs for presentation to cells of the immune system whilesimultaneously stimulating a strong antigen-specific immune response. Insome aspects of the invention, the antigen is a lipopeptide.

U.S. Pat. No. 7,303,881, incorporated by reference herein in itsentirety, discloses that multiple cationic lipids complexed withdisease-associated antigens were shown to stimulate a prophylacticimmune response that prevented the specific disease (e.g., HPV-positivecancer) and also a therapeutic immune response that killed cellsexpressing the particular antigen and resulted in an effective treatmentof the disease. Presently, studies were performed to further understandthe effects of chirality on the immunostimulatory capability of cationiclipids by using the R and S enantiomers of DOTAP. (The R and Senantiomers of DOTAP are shown in FIGS. I A and 113). These studies haveled to the discovery that individual enantiomers of cationic lipids canfunction independently as immunodulators to stimulate an immune responsewith (or without) antigens. Further, when enantiomers of cationic lipidsare complexed with an antigen, an antigen specific immune response isgenerated. The extent of the disease specific immune response differssignificantly between the R and S enantiomers of the cationic lipid.

In another aspect, the chiral cationic lipid, at a dose sufficient tostimulate an immune response, is administered in combination with anantigen or antigens. In this case the cationic lipid/antigen combinationis capable of generating an immune response that is specific to theantigen(s) delivered in combination with the cationic lipid. Theresponse generated may include production of specific cytotoxic T cells,memory T cells, or B cells resulting in the prevention of, ortherapeutic response to, the specific disease associated with theantigen(s).

The chiral cationic lipids of the invention may be in the form ofcationic lipid complexes. The cationic lipid complex can take the formof various vesicles such as liposomes, micelles, or emulsions. Thecationic lipid complexes may be unilaminar or multilaminar. When anantigen is included, the antigen may be encapsulated in the cationiclipid complex or may be unencapsulated. Encapsulated is understood tomean that the antigen may be contained within the internal space of thecomplex and/or incorporated into the lipid walls of the complex.

Another aspect of the invention relates to a method for producing thesecomplexes, wherein the method may optionally include the step ofpurifying these formulations from excess individual components.

In certain embodiments, the cationic lipid complexes have a net positivecharge and/or a positively charged surface at pH 6.0-8.0.

The optional “antigen” which may be included with cationic lipidcomplexes of the invention may be nucleic acids, peptides, lipopeptides,proteins, lipoproteins, polysaccharides, and other macromolecules whichmay be complexed directly with cationic lipids. However, cationic drugs(e.g., large cationic protein) can be directly complexed with an anioniclipid or sequentially complexed first with anionic lipid or polymerfollowed by the chiral cationic lipid. The use of this process permitsdelivery of positive or neutral charged drugs to cells by the complexesof the present invention.

One aspect of the present invention involves the use of the chiralcationic lipid complexes to activate dendritic cells and also tostimulate the production of chemokines and cytokines. Chemokines andcytokines are important regulators of immune responses. Chemokines wereoriginally identified as potent chemoattractants for inflammatory cellsincluding neutrophils, eosinophils, and in onocytes/macrophages.Subsequent studies have revealed that chemokines have profound effectson immune reactions by regulating the trafficking of dendritic cells andother lymphocytes into lymphoid organs. Dendritic cells are migratorycells that sample antigens in the tissue, migrate to the draining lymphnodes and mature to stimulate the T cell response. CCL2, a member of theCC chemolcines was originally identified as a chemotactic and activatingfactor for monocytes/macrophages. Subsequent studies showed that it canalso affect the function of T cells, natural killer cells, andneutrophils. Further exploration found that CCL2 was the most potentactivator of CD8+ cytotoxic T lymphocytes (“CTL”) activity, when in thepresence of the Thl cytokines, interleukin-12 (“IL-12”) and interferon-y(IFN-y”). This can be explained by a positive bidirectional interactionbetween CCL2 and IFN-y systems. An absence of either the cytokine orchemokine may interfere with Thl polarization and subsequent specifictumor immunity generation. Another CC chemokine, CCL-4, has also beenshown to recruit and expand dendritic cells in vivo and potentiate theimmunogenicity of plasmid DNA vaccines. Recently, it has been shown thatchemokines enhance immunity by guiding naive CDS+ T cells to sites ofCD4+ T cell-dendritic cell interaction and promote memory CD8+ T cellgeneration. A few examples of chemokines that may be stimulated by thecationic lipid complexes of the present invention are CCL-2, CCL-3, andCCL-4. Examples of cytokines that may be stimulated by the cationiclipid complexes of the present invention are IL-2, IL-8, IL-12 andIFN-y. The inventors contemplate that the cationic lipid complexes ofthe present invention may stimulate chemokines and cytokines in additionto those disclosed in this specification.

Lipids

The chiral cationic lipid complexes of the present invention may formliposomes that are optionally mixed with antigen and may contain thechiral cationic lipids alone or chiral cationic lipids in combinationwith neutral lipids. Suitable chiral cationic lipid species include, butare not limited to the R and S enantiomers of: 3-β[⁴N-(¹N,⁸-diguanidinospennidine)-carbaroyl]cholesterol (BGSC);3-β0[N,N-diguanidinoethyl-aminoethane)-carbamoyl] cholesterol (BGTC);N,N¹N²N³Tetra-methyttetrapalmitylsperrnine (cellfectin);N-t-butyl-N′-tctraclecyl-3-tetradecyl-aininopropion-amidine(CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB);1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide(DMRIE);2,3-dioleoyloxy-N-[2(spenninecarboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoracetate) (DOSPA); 1,3-dioleoyloxy-2-(6-carboxyspennyl)-propylamide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole(DPIM) N,N,1\1¹,Ni-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3dioleoyloxy-1,4-butanediammonium iodide) (Tfx-50); N-I-(2,3-dioleoyloxy)propyl-N,N,N-trimethyl ammonium chloride (DOTMA) or otherN—(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted. ammonium surfactants; 1,2dioleoyl-3-(4′-trimethylammonio) butanol-sn-glycerol (DOBT) orcholesteryl (4′trimethylammonia) butanoate (ChOTB) where the trimethylammonium group is connected via a butanol spacer arm to either thedouble chain (for DOTB) or cholesteryl group (for ChOTB); DORI(DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE(DL-1,2-O-dioleoyl-3-dimethylaminopropyl-(3-hydroxyethylammonium)(DORIE) or analogs thereof as disclosed in WO 93/03709;2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesterylhemisuccinate ester (ChOSC); lipopolyamines such asdioctadecylamidoglycylspermine (DOGS) and dipalinitoylphospliatidylethanolamylspennine (DPPES) or the cationic lipidsdisclosed in U.S. Pat. No. 5,283,185, cholesteryl-3β-carboxyl-amido-ethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylateiodide, cholesteryl-3-O-carboxyamidoethyleneamine,cholesteryl-3-β-oxysuccinamido-ethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxosuccinateiodide, 2-(2-trimethylammonio)-ethylmethylaminoethyl-cholesteryl-3-β-oxysuccinate iodide,3-β-N-(1\1′,N′-dimethylaminoethane) carbamoyl cholesterol (DC-chol), and3-β-N-(polyethyleneimine)-carbamoylcholesterol; 0,0′-dirnyristyl-N-lysylaspartate (DMKE); 0,0′-dirnyristyl-N-lysyl-glutamate (DMKD);1,2-climyristyloxypropyl-3-dintethyl-hydroxy ethyl ammonium bromide(DMRIE); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DMEPC);1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC);1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC);1,2-clipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC);1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DMEPC);1,2-dioleoyl-3-trimethylammonium propane (DOTAP); dioleoyl dimethylaminopropane (DODAP); 1,2-pahnitoyl-3-trimethylammonium propane(DSTAP); 1,2-distearoyl-3-trimetbylam onium propane (DSTAP),1,2-nlyristoyl-3-trimethylammonium propane (DMTAP); and sodium dodecylsulfate (SDS). The present invention contemplates the use of structuralvariants and derivatives of the cationic lipids disclosed in thisapplication.

Certain aspects of the present invention include nonsteroidal chiralcationic lipids having a structure represented by the following formula:

wherein in R³ is a quaternary ammonium group, Y¹ is chosen from ahydrocarbon chain, an ester, a ketone, and a peptide, C* is a chiralcarbon, R² and R³ are independently chosen from a saturated fatty acid,an unsaturated fatty acid, an ester-linked hydrocarbon,phosphor-diesters, and combinations thereof. DOTAP, DMTAP, DSTAP, DPTAP,DPEPC, DSEPC, DMEPC, DLEPC, DOEPC, DMKE, DMKD, DOSPA, DOTMA, areexamples of lipids having this general structure.

In one embodiment, chiral cationic lipids of the invention are lipids inwhich bonds between the lipophilic group and the amino group are stablein aqueous solution. Thus, an attribute of the complexes of theinvention is their stability during storage (i.e., their ability tomaintain a small diameter and retain biological activity over timefollowing their formation). Such bonds used in the cationic lipidsinclude amide bonds, ester bonds, ether bonds and carbamoyl bonds. Thoseof skill in the art would readily understand that liposomes containingmore than one cationic lipid species maybe used to produce the complexesof the present invention. For example, liposomes comprising two cationiclipid species, lysyl-phosphatidylethanolamine and β-alanyl cholesterolester have been disclosed for certain drug delivery applications[Brunette, E. et al., Nucl. Acids Res., 20:1151 (1992)].

It is to be further understood that in considering chiral cationicliposomes suitable for use in the invention and optionally mixing withantigen, the methods of the invention are not restricted only to the useof the cationic lipids recited above but rather, any lipid compositionmay be used so long as a cationic liposome is produced and the resultingcationic charge density is sufficient to activate and induce an immuneresponse.

Thus, the complexes of the invention may contain other lipids inaddition to the chiral cationic lipids, These lipids include, but arenot limited to, lyso lipids of which lysophosphatidylcholine (1-oleoyllysophospliatidyleholine) is an example, cholesterol, or neutralphospholipids including dioleoyl phosphatidyl ethanolamine (DOPE) ordioleoyl phosphatidylcholine (DOPC) as well as various lipophilicsurfactants, containing polyethylene glycol moieties, of which Tween-80and PEG-PE are examples.

The chiral cationic lipid complexes of the invention may also containnegatively charged lipids as well as cationic lipids so long as the netcharge of the complexes formed is positive and/or the surface of thecomplex is positively charged. Negatively charged lipids of theinvention are those comprising at least one lipid species having a netnegative charge at or near physiological pH or combinations of these.Suitable negatively charged lipid species include, but are not limitedto, CHEMS (cholesteryl hemisuccinate), NGPE (N-glutarylphosphatidlylethanolanine), phosphatidyl glycerol and phosphatidic acidor a similar phospholipid analog.

Methods for producing the liposomes to be used in the production of thelipid comprising drug delivery complexes of the present invention areknown to those of ordinary skill in the art. A review of methodologiesof liposome preparation may be found in Liposome Technology (CFC PressNew York 1984); Liposomes by Ostro (Marcel Dekker, 1987); MethodsBiochem Anal, 33:337-462 (1988) and U.S. Pat. No. 5,283,185. Suchmethods include freeze-thaw extrusion and sonication. Both unilamellarliposomes (less than about 200 mu in average diameter) and multilamellarliposomes (greater than about 300 nm in average diameter) may be used asstarting components to produce the complexes of this invention.

In the cationic liposomes utilized to produce the cationic lipidcomplexes of this invention, the chiral cationic lipid is present in theliposome at from about 10 mole % to about 100 mole % of total liposomallipid, or from about 20 mole % to about 80 mole %. The neutral lipid,when included in the liposome, may be present at a concentration of fromabout 0 mole % to about 90 mole % of the total liposomal lipid, or fromabout 20 mole % to about 80 mole %, or from 40 mole % to 80 mole %. Thenegatively charged lipid, when included in the liposome, may be presentat a concentration ranging from about 0 mole % to about 49 mole % of thetotal liposomal lipid, or from about 0 mole % to about 40 mole %. In oneembodiment, the liposomes contain a chiral cationic and a neutral lipid,in ratios between about 2:8 to about 6:4.

It is further understood that the complexes of the present invention maycontain modified lipids, protein, polycations or receptor ligands whichfunction as a targeting factor directing the complex to a particulartissue or cell type. Examples of targeting factors include, but are notlimited to, asialoglycoprotein, insulin, low density lipoprotein (LDL),folate and monoclonal and polyclonal antibodies directed against cellsurface molecules. Furthermore, to modify the circulatory half-life ofthe complexes, the positive surface charge can be sterically shielded byincorporating lipophilic surfactants which contain polyethylene glycolmoieties.

The cationic lipid complexes may be stored in isotonic sucrose ordextrose solution upon collection from the sucrose gradient or they maybe lyophilized and then reconstituted in an isotonic solution prior touse. In one embodiment, the cationic lipid complexes are stored insolution. The stability of the cationic lipid complexes of the presentinvention is measured by specific assays to determine the physicalstability and biological activity of the cationic lipid complexes overtime in storage. The physical stability of the cationic lipid complexesis measured by determining the diameter and charge of the cationic lipidcomplexes by methods known to those of ordinary skill in the art,including for example, electron microscopy, gel filtrationchromatography or by means of quasi-elastic light scattering using, forexample, a Coulter N4SD particle size analyzer as described in theExample, The physical stability of the cationic lipid complex is“substantially unchanged” over storage when the diameter of the storedcationic lipid complexes is not increased by more than 100%, or by notmore than 50%, or by not more than 30%, over the diameter of thecationic lipid complexes as determined at the time the cationic lipidcomplexes were purified.

While it is possible for the chiral cationic lipid to be administered ina pure or substantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation. Pharmaceuticalformulations using the chiral cationic lipid complexes of the inventionmay comprise the cationic lipid complexes in a physiologicallycompatible sterile buffer such as, for example, phosphate bufferedsaline, isotonic saline or low ionic strength buffer such as acetate orHepes (an exemplary pH being in the range of about 3.0 to about 8.0).The chiral cationic lipid complexes may be administered as aerosols oras liquid solutions for intratumoral, intraarterial, intravenous,intratracheal, intraperitoneal, subcutaneous, and intramuscularadministration.

The formulations of the present invention may incorporate any stabilizerknown in the art. Illustrative stabilizers arc cholesterol and othersterols that may help rigidify the liposome bilayer and preventdisintegration or destabilization of the bilayer. Also agents such aspolyethylene glycol, poly-, and monosaccharides may be incorporated intothe liposome to modify the liposome surface and prevent it from beingdestabilized due to interaction with blood-components. Otherillustrative stabilizers are proteins, saccharides, inorganic acids, ororganic acids which may be used either on their own or as admixtures.

A number of pharmaceutical methods may be employed to control, modify,or prolong the duration of immune stimulation. Controlled releasepreparations may be achieved through the use of polymer complexes suchas polyesters, polyamino acids, methylcellulose, polyvinyl, poly(lacticacid), and hydrogels to encapsulate or entrap the cationic lipids andslowly release them. Similar polymers may also be used to adsorb theliposomes. The liposomes may be contained in emulsion formulations inorder to alter the release profile of the stimulant. Alternatively, theduration of the stimulant's presence in the blood circulation may beenhanced by coating the surface of the liposome with compounds such aspolyethylene glycol or other polymers and other substances such assaccharides which are capable of enhancing the circulation time or halflife of liposomes and emulsions.

When oral preparations are required, the chiral cationic lipids may becombined with typical pharmaceutical carriers known in the art such as,for example, sucrose, lactose, methylcellulose, carboxymethyl cellulose,or gum Arabic, among others. The cationic lipids may also beencapsulated in capsules or tablets for systemic delivery.

Administration of the chiral cationic lipid of the present invention maybe for either a prophylactic or therapeutic purpose. When providedprophylactically, the cationic lipid is provided in advance of anyevidence or symptoms of illness. When provided therapeutically, thecationic lipid is provided at or after the onset of disease. Thetherapeutic administration of the immune-stimulant serves to attenuateor cure the disease. For both purposes, the cationic lipid may beadministered with an additional therapeutic agent(s) or antigen(s). Whenthe cationic lipids are administered with an additional therapeuticagent or antigen, the prophylactic or therapeutic effect may begenerated against a specific disease.

The formulations of the present invention, both for veterinary and forhuman use, comprise a chiral cationic lipid alone as described above, asa mixture of R and S enantiomers, or also optionally, with one or moretherapeutic ingredients such as an antigen(s) or drug molecule(s). Theformulations may conveniently be presented in unit dosage form and maybe prepared by any method known in the pharmaceutical art.

Antigens

In one embodiment, the chiral cationic lipid is administered without anyadditional agents in order to boost or lower various immune responses,including production of other immune modulators, and to boost the immuneresponse to fighting disease. In another embodiment, the chiral cationiclipid is administered in combination with an antigen or antigens. Inthis case the objective is to generate an immune response, which isspecific to the antigen(s) delivered in combination with the cationiclipid. The response generated may include production of specificcytotoxic T-cells, memory T-cells, or B-cells resulting in theprevention of or therapeutic response to the specific disease associatedwith those antigen(s). The antigen can be any tumor-associated antigenor microbial antigen or any other antigen known to one skilled in theart.

A “tumor-associated antigen,” as used herein is a molecule or compound(e.g., a protein, peptide, polypeptide, lipoprotein, lipopeptide,glycoprotein, glycopeptides, lipid, glycolipid, carbohydrate, RNA,and/or DNA) associated with a tumor or cancer cell and which is capableof provoking an immune response (humoral and/or cellular) when expressedon the surface of an antigen presenting cell in the context of a majorhistocompatibility complex (“MHC”) molecule. Tumor-associated antigensinclude self antigens, as well as other antigens that may not bespecifically associated with a cancer, but nonetheless enhance an immuneresponse to and/or reduce the growth of a tumor or cancer cell whenadministered to an animal. More specific embodiments are providedherein.

A “microbial antigen,” as used herein, is an antigen of a microorganismand includes, but is not limited to, infectious virus, infectiousbacteria, infectious parasites and infectious fungi. Microbial antigensmay be intact microorganisms, and natural isolates, fragments, orderivatives thereof, synthetic compounds which are identical to orsimilar to naturally-occurring microbial antigens and, preferably,induce an immune response specific for the corresponding microorganism(from which the naturally-occurring microbial antigen originated). In apreferred embodiment, a compound is similar to a naturally-occurringmicroorganism antigen if it induces an immune response (humoral and/orcellular) similar to a naturally-occurring microorganism antigen.Compounds or antigens that are similar to a naturally-occurringmicroorganism antigen are well known to those of ordinary skill in theart such as, for example, a protein, peptide, polypeptide, Lipoprotein,lipopeptide, glycoprotein, glycopeptides, lipid, glycolipid,carbohydrate, RNA, and/or DNA. Another nonlimiting example of a compoundthat is similar to a naturally-occurring microorganism antigen is apeptide mimic of a polysaccharide antigen. More specific embodiments areprovided herein.

The term “antigen” is further intended to encompass peptide or proteinanalogs of known or wild-type antigens such as those described in. thisspecification. The analogs may be more soluble or more stable than wildtype antigen, and may also contain mutations or modifications renderingthe antigen more immunologically active. Antigen can be modified in anymanner, such as adding lipid or sugar moieties, mutating peptide orprotein amino acid sequences, mutating the DNA or RNA sequence, or anyother modification known to one skilled in the art. Antigens can bemodified using standard methods known by one skilled in the art.

Also useful in the compositions and methods of the present invention arepeptides or proteins which have amino acid sequences homologous with adesired antigen's amino acid sequence, where the homologous antigeninduces an immune response to the respective tumor, microorganism orinfected cell.

In one embodiment, the antigen in the cationic lipid complex comprisesan antigen associated with a tumor or cancer, i.e., a tumor-associatedantigen, to make a vaccine to prevent or treat a tumor. As such, in oneembodiment, the tumor or cancer vaccines of the present inventionfurther comprise at least one epitope of at least one tumor-associatedantigen. In another preferred embodiment, the tumor or cancer vaccinesof the present invention further comprise a plurality of epitopes fromone or more tumor-associated antigens. The tumor-associated antigensfinding use in the cationic lipid complexes and methods of the presentinvention can be inherently immunogenic, or nonimmunogenic, or slightlyimmunogenic. As demonstrated herein, even tumor-associated self antigensmaybe advantageously employed in the subject vaccines for therapeuticeffect, since the subject compositions are capable of breaking immunetolerance against such antigens. Exemplary antigens include, but are notlimited to, synthetic, recombinant, foreign, or homologous antigens, andantigenic materials may include but are not limited to proteins,peptides, polypeptides, lipoproteins, lipopeptides, lipids, glycolipids,carbohydrates, RNA and DNA. Examples of such vaccines include, but arenot limited to, those for the treatment or prevention of breast cancer,head and neck cancer, melanoma, cervical cancer, Lung cancer, prostatecancer, gut carcinoma, or any other cancer known in the art using acationic lipid in a complex with a tumor-associated antigen(s), It isalso possible to formulate the antigen. with the cationic lipid withoutencapsulating it in the liposome. Thus, the chiral cationic lipidcomplexes of the present invention may be used in methods to treat orprevent cancer. In. such a case, the mammal to be immunized maybeinjected with the pharmaceutical formulation containing the liposomewith the encapsulated antigen(s).

Tumor-associated antigens suitable for use in the present inventioninclude both naturally occurring and modified molecules which may beindicative of single tumor type, shared among several types of tumors,and/or exclusively expressed or overexpressed in tumor cells incomparison with normal cells. In addition to proteins, glycoproteins,lipoproteins, peptides, and lipopeptides, tumor-specific patterns ofexpression of carbohydrates, gangliosides, glycolipids, and mucins havealso been documented. Exemplary tumor-associated antigens for use incancer vaccines include protein products of oncogenes, tumor suppressorgenes, and other genes with mutations or rearrangements unique to tumorcells, reactivated embryonic gene products, oncofetal antigens,tissue-specific (but not tumor-specific) differentiation antigens,growth factor receptors, cell surface carbohydrate residues, foreignviral proteins, and a number of other self proteins.

Specific embodiments of tumor-associated antigens include, e.g., mutatedor modified antigens such as the protein products of the Ras p21protooncogenes, tumor suppressor p53 and HER-2/neu and BCR-abloncogenes, as well as CDK4, MUM1, Caspase 8, and Beta catenin;overexpressed antigens such as galectin 4, galectin 9, carbonicanhydrase, Aldolase A, PRAMS, Her2/neu, ErbB-2 and KSA, oncofetalantigens such as alpha fetoprotein (AFP), human chorionic gonadotropin(hCG); self antigens such as carcinoembryonic antigen (CEA) andmelanocyte differentiation antigens such as Mart I/Melan A, 0)100, gp75,Tyrosinase, TRP I and TRP2; prostate associated antigens such as PSA,PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene products suchas MA.GE 1, MAGE 3, MACE 4, GAGE 1, GAGE 2, BAGE, RAGE, and other cancertestis antigens such as NY-ES01, SSX2 and SCP1; mucins such as Muc-1 andMuc-2; gangliosides such as GM2, GD2 and GD3, neutral glycolipids andglycoproteins such as Lewis (y) and globo-H; and glycoproteins such asTo, Thompson-Freidenreich antigen (TF) and sTn. Also included astumor-associated antigens herein are whole cell and tumor cell lysatesas well as immunogenic portions thereof, as well as immunoglobulinidiotypes expressed on monoclonal proliferations of B lymphocytes foruse against B cell lymphomas.

Tumor-associated antigens and their respective tumor cell targetsinclude, e.g., cytokeratins, particularly cytokeratin 8, 18 and 19, asantigens for carcinoma. Epithelial membrane antigen (EMA), humanembryonic antigen (HEA-125), human milk fat globules, MBrl, MBr8,Ber-EP4, 17-1 A, C26 and T16 are also known carcinoma antigens. Desminand muscle-specific actin are antigens of cryogenic sarcomas. Placentalalkaline phosphatase, beta-human chorionic gonadotropin, andalpha-fetoprotein are antigens of trophoblastic and germ cell tumors.Prostate specific antigen is an antigen of prostatic carcinomas,carcinoembryonic antigen of colon adenocarcinomas. EIMB-45 is an antigenof melanomas. In cervical cancer, useful antigens could be encoded byhuman papilloma virus. Chromagranin-A and synaptophysin are antigens ofneu pendocrine and neuroectodermal tumors. Of particular interest areaggressive tumors that form solid tumor masses having necrotic areas.The lysis of such necrotic cells is a rich source of antigens forantigen-presenting cells, and thus the subject therapy may findadvantageous use in conjunction with conventional chemotherapy and/orradiation therapy.

In one embodiment, the human papillomavirus HPV antigens are used. Aspecific HPV antigen that used as a tumor-associated antigen is HPVsubtype 16 E7. HPV E7 antigen-cationic lipid complexes are effective atpreventing and treating cervical cancer. In addition, a geneticallyengineered E7 protein, i.e., E7 in protein, having antigenic activity,but without tumorigenic activity, is an effective tumor-associatedantigen. Elm-cationic lipid complexes induce cellular immunity to causecomplete regression of established tumors and, thus, are useful aspotent anti-cervical cancer vaccines.

Tumor-associated antigens can be prepared by methods well known in theart. For example, these antigens can be prepared from cancer cellseither by preparing crude extracts of cancer cells (e.g., as describedin Cohen et al., Cancer Res., 54:1055 (1994)), by partially purifyingthe antigens, by recombinant technology, or by de novo synthesis ofknown antigens. The antigen may also be in the form of a nucleic acidencoding an antigenic peptide in a form suitable for expression in asubject and presentation to the immune system of the immunized subject.Further, the antigen may be a complete antigen, or it may be a fragmentof a complete antigen comprising at least one epitope.

Antigens derived from pathogens known to predispose to certain cancersmay also be advantageously included in the cancer vaccines of thepresent invention. It is estimated that close to 16% of the worldwideincidence of cancer can be attributed to infectious pathogens; and anumber of common malignancies are characterized by the expression ofspecific viral gene products. Thus, the inclusion of one or moreantigens from pathogens implicated in causing cancer may help broadenthe host immune response and enhance the prophylactic or therapeuticeffect of the cancer vaccine. Pathogens of particular interest for usein the cancer vaccines provided herein include the, hepatitis B virus(hepatocellular carcinoma), hepatitis C virus (heptomas), Epstein Barrvirus (EMT) (Burkitt lymphoma, nasopharynx cancer, PTLD inimmunosuppressed individuals), I-ITLVL (adult T cell leukemia),oncogenic human papilloma viruses types 16, 18, 33, 45 (adult cervicalcancer), and the bacterium Helicobacter pylori (B cell gastriclymphoma). Other medically relevant microorganisms that may serve asantigens in mammals and more particularly humans are describedextensively in the literature, e.g., C. G. A Thomas, MedicalMicrobiology, Baiiliero Tindall, Great Britain 1983, the entire contentsof which is hereby incorporated by reference.

In another embodiment, the antigen in the cationic lipid complexcomprises an antigen derived from or associated with a pathogen, i.e., amicrobial antigen. As such, in one embodiment, the pathogen vaccines ofthe present invention further comprise at least one epitope of at leastone microbial antigen. Pathogens that may be targeted by the subjectvaccines include, but are not limited to, viruses, bacteria, parasitesand fungi. In another embodiment, the pathogen vaccines of the presentinvention further comprise a plurality of epitopes from one or moremicrobial antigens.

The microbial antigens finding use in the cationic lipid complexes andmethods may be inherently immunogenic, or nonimmunogenic, or slightlyimmunogenic. Exemplary antigens include, but are not limited to,synthetic, recombinant, foreign, or homologous antigens, and antigenicmaterials may include but are not limited to proteins, peptides,polypeptides, lipoproteins, lipopeptides, lipids, glycolipids,carbohydrates, RNA, and DNA.

Exemplary viral pathogens include, but are not limited to, viruses thatinfect mammals, and more particularly humans. Examples of virus include,but are not limited to: Retroviridae (e.g., human immunodeficiencyviruses), such as HIV-1 (also referred to as HTLV-III, LAV orHTLV-IHiLAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridac (e.g. polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.strains that cause gastroenteritis); Togavridae (e.g. equineencephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses,encephalitis viruses, yellow fever viruses); Coronoviridae (e.g.coronaviruses); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabiesviruses); Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g.vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebolaviruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses,phleboviruses and Naito viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); andlridoviridae (e.g. African swine fever virus); and unclassified viruses(e.g. the etiological agents of Spongiform encephalopathies, the agentof delta hepatitis (thought to be a defective satellite of hepatitis Bvirus), the agents of non-A, non-B hepatitis (class 1.⁻-internallytransmitted; class 2=parenterally transmitted (i.e. Hepatitis C);Norwalk and related viruses, and astroviruses).

Also, gram negative and grain positive bacteria may be targeted by thesubject compositions and methods in vertebrate animals. Such grainpositive bacteria include, but are not limited to Pasteurelkt species,Staphylococci species, and Streptococcus species. Gram negative bacteriainclude, but are not limited to, Escherich fa coli, Pseudomonas species,and Salmonella species. Specific examples of infectious bacteria includebut are not limited to: Helicobacter pyloris, Borella burgdoiferi,Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M.avium, M. intracellulare, M. kansaii, M., gordonae), Staphylococcusaureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeriamonocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneuinoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus infitenzae, Bacillusantracis, Corynebacterium diphtheriae, coiynebacteriuin sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumletani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira,Rickettsia, and Actinoinyces.

Polypeptides of bacterial pathogens which may find use as sources ofmicrobial antigens in the subject compositions include but are notlimited to an iron-regulated outer membrane protein, (“TROMP”), an outermembrane protein (“OMP”), and an A-protein of A eromonis sahnonicidawhich causes furunculosis, p57 protein of Renibacterium salmonittarumwhich causes bacterial kidney disease (“BKD”), major surface associatedantigen (“msa”), a surface expressed cytotoxin (“mpr”), a surfaceexpressed hemolysin (“ish”), and a flagellar antigen of Yersiniosis; anextracellular protein (“ECP”), an iron-regulated outer membrane protein(“TROMP”), and a structural protein of Pasteurellosis; an OMP and aflagellar protein of Vibrosis anguillarum and V ordalii; a flagellarprotein, an OMP protein, aroA, and purA of Edwardsiellosis ictaluri andE, tarda; and surface antigen of chthyophthirius; and a structural andregulatory protein of Cytophaga columnari; and a structural andrectulatory protein of Rickettsia. Such antigens can be isolated orprepared recombinantly or by any other means known in the art.

Examples of pathogens further include, but are not limited to, fungithat infect mammals, and more particularly humans. Examples of fungiinclude, but are not limited to: Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, and Candida albicans. ‘Examples of infectious parasitesinclude Plasmodium such as Plasmodium. falciparum, Plasmodium malartae,Plasmodium ovale, and Plasmodium vivax. Other infectious organisms (i.e.protists) include Toxoplasma gondii. Polypeptides of a parasiticpathogen include but are not limited to the surface antigens ofkhthyophthirius.

Other medically relevant microorganisms that serve as antigens inmammals and more particularly humans are described extensively in theliterature (e.g., see C. G. A Thomas, Medical Microbiology, BailliereTindall, Great Britain 1983). In addition to the treatment of infectioushuman diseases and human pathogens, the compositions and methods of thepresent invention are useful for treating infections of nonhumanmammals. Many vaccines for the treatment of nonhuman mammals aredisclosed in Bennett, K. Compendium of Veterinary Products, 3rd ed.North American Compendiums, Inc., 1995; see also WO 02/069369, thedisclosure of which is expressly incorporated by reference herein in itsentirety.

Exemplary nonhuman pathogens include, but are not limited to, mousemammary tumor virus (“MMTV”), Rous sarcoma virus (“RSV”), avian leukemiavirus (“ALV”), avian myeloblastosis virus (“AMV”), murine leukemia virus(“MLV”), feline leukemia virus (“FeLV”), murine sarcoma virus (“MSV”),gibbon ape leukemia virus (“GALV”), spleen necrosis virus (“SNV”),reticuloendotheliosis virus (“RV”), simian sarcoma virus (“SSV”),Mason-Pfizer monkey virus (“MPMV”), simian retrovirus type 1 (“SRV-1”),lentiviruses such as HIV-I, HIV-2, SIV, Visna virus, felineimmunodeficiency virus (“Fly”), and equine infectious anemia virus(“EIAY”), T-cell leukemia viruses such as HTLV-1, HTLY-II, simian T-cellleukemia virus (“STLV”), and bovine leukemia virus (“III,V”), and foamyviruses such as human foamy virus (“HFV”), simian foamy virus (“SFV”)and bovine foamy virus (“B1⁷V”).

In some embodiments, “treatment,” “treat,” and “treating,” as usedherein with reference to infectious pathogens, refer to a prophylactictreatment which increases the resistance of a subject to infection witha pathogen or decreases the likelihood that the subject will becomeinfected with the pathogen; and/or treatment after the subject hasbecome infected in order to fight the infection, e.g., reduce oreliminate the infection or prevent it from becoming worse.

Microbial antigens can be prepared by methods well known in the art. Forexample, these antigens can be prepared directly from viral andbacterial cells either by preparing crude extracts, by partiallypurifying the antigens, or alternatively by recombinant technology or byde MVO synthesis of known antigens. The antigen may also be in the formof a nucleic acid encoding an antigenic peptide in a form suitable forexpression in a subject and presentation to the immune system of theimmunized subject. Further, the antigen may be a complete antigen, or itmay be a fragment of a complete antigen comprising at least one epitope.

In order to improve incorporation of the antigen into the chiralcationic lipid vesicles and also to improve delivery to the cells of theimmune system, the antigen may be conjugated to a lipid chain in orderto improve its solubility in the hydrophobic acyl chains of the cationiclipid, while maintaining the antigenic properties of the molecule. Thelipidated antigen can be a lipoprotein, or a lipopeptide, andcombinations thereof. The lapidated antigen may have a linker conjugatedbetween the lipid and the antigen such as, for example, an N-terminal aor a-palmitoyl lysine may be connected to antigen via a dipeptideSer-Ser linker. U.S. application Ser. No. 12/049,957 discloses that theDOTAP/E7-lipopeptide complex exhibited an enhanced functionalantigen-specific CD8⁻ T lymphocyte responses in vivo compared to theDOTAP/E7 formulation, and therefore provided superior anti-tumorefficacy.

The present invention will be further appreciated in light of thefollowing example.

Example

Effective Stimulation of the Immune System by Enantiomers of CationicLipids

I. Cell Lines and Peptides

TC-1 cells are C57BL/6 mouse lung endothelial cells that have beentransformed with the HPV16 E6 and E7 oncogenes and activated H-ras.Cells were grown in RPMI medium (commercially available from Invitrogenof Carlsbad, Calif.) supplemented with 10% fetal bovine serum and 100U/mI penicillin, and 100 mg/ml streptomycin. The MHC class I restrictedpeptide from the HPV 16 E7 protein (amino acid 11 to 20, YMLDLQPETT[SEQ. ID. NO. I]) was synthesized by the University of PittsburghPeptide Synthesis Facility by solid state synthesis using an AdvancedChernTech model 200 peptide synthesizer and purified by HPLC.

2. Preparation of Lipid/Antigen Complexes and Determination of PhysicalProperties

The enantiomers of DOTAP were supplied by Merck AG (EPROVA),Switzerland. All other lipids were purchased from Avanti Polar Lipids(Alabaster, Ala.). Small unilamellar DOTAP liposomes were prepared bythin film hydration followed by extrusion. The lipid, in chloroform, wasdried as a thin layer under a stream of nitrogen in a glass tube. Thethin film was vacuum desiccated for 2-3 h and then re-hydrated in cellculture grade water (commercially available from Cambrex ofWalkersville, Md.) or buffer (such buffers are well known to thoseskilled in the art) containing E7 peptide to a final concentration of0.7 mg lipids and 0.1 mg E7 per mL (molar ratio=11:1). The lipiddispersion was sequentially extruded through polycarbonate membraneswith pore size of 0.4, 0.2, and 0.1 pan. The un-entrapped peptide wasnot removed. The liposomes were stored at 4° C. until use. E7 peptideassociation with the liposome was determined by measuring the percentageof liposome-bound peptide. In brief, unbound E7 peptide from R-DOTAP/E7,S-DOTAP/E7 or RS-DOTAP/E7 complexes was separated by a Microcon®centrifugal filtrate device (Millipore, Bedford, Mass.) and theconcentration of unbound peptide was measured by Micro BCA™ ProteinAssay Kit (Pierce, Rockford, Ill.). The efficiency of peptideassociation was determined as percent unbound peptide. Other methodsused in general liposome preparation that are well known to thoseskilled in the art may also be used.

3. Statistical Analysis

Data are presented as mean±SD of at least 3 independent experiments.Two-tailed Student's t tests were used to assess statisticalsignificance for differences in means. Significance was set at p<0.05.

4. Individual R and S Enantiorners of Cationic Lipid/E7 ComplexesActivate Human Dendritic Cells Similarly to the DOATP Racemic Mixture.

Cationic liposomes were prepared as described above. The E7 antigen usedin the formulation is the identified human E7 peptide restricted byHLA-A*0201 [HPV-16 E7, amino acids 11-20, YMLDLQPETT (SEQ. ID. NO. 1)1The peptide was synthesized by the University of Pittsburgh, MolecularMedicine Institute, Pittsburgh, Pa. Human 1-ILA-A2 human dendritic cellswere obtained from Lonza (of Walkersville, Md.). Frozen cryovials werethawed and the dendritic cells were cultured in LGM-3 medium(commercially available from Lonza of Walkersville, Md.) supplementedwith 50 microgram/mI IL-4 and GM-CSF at 37° C. and 5% CO₂ at an initialplating density of 125,000 cells/cm² in 2 ml of medium in 12-well tissueculture dishes. The cells were grown for 3 days in culture and appearedas a mixture of adherent and rounded cells by microscopic examination.

The cells were treated on day 3 with a fresh dose of 50 microgram/ml ofIL-4 and GM-CSF (all wells) and test wells were treated with either amixture of interleukin 1-beta (“IL-β”), interleukin 6 (“IL-6”) and TNF-aat 10 ng/ml, and prostaglandin E2 (“PGE-2”) at 10 μg/ml (positivecontrol for activation), no treatment (negative activation control), andS-DOTAP/E7 at 2.5, 10 and 40 micromolar final concentrations, andR-DOTAP/E7 at 2.5, 10 and 40 micromolar final concentrations. Thetreated dendritic cells were maintained in culture for 24 hours andharvested for cell surface marker staining and flow cytornetry analysis.The harvested cells were counted by hernacytometer and 10 pi of thefollowing antibody conjugates were added sequentially to each sample forlabeling surface markers: CD80-FITC, CD83-APC, and CD86-PE (BDBiosciences). The surface labeled cells were subsequently analyzed byflow cytometry using a BD FACxcaliber flow cytometer, and theco-stimulatory dendritic cell marker molecules CD80, CD83, and CD86which are produced upon activation, were monitored. As seen in FIGS. 2,3 and 4 primary human dendritic cells treated with the both enantiomersof the cationic lipid/E7 complex up-regulated the expression of allthree co-stimulatory markers of dendritic cell activation evaluated andrequired for successful antigen presentation to T-cells, similarly towhat was observed with the racemic mixture (RS-DOTAP) of the cationiclipid and reported in U.S. application Ser. No. 12/049,957, assigned tothe assignee of the present application.

5. Cationic Lipid/E7 Complexes Consisting of Individual R and SEnantiomers Exhibit Different Potencies in Activating Human DendriticCells to Induce Chemokine and Cytokine Production

Human HLA-A 2 dendritic cells (Lonza, Walkersville, Md.), were treatedand grown in culture as described above. On day 3 the cells were treatedwith 40 micromolar DOTAP/E7 complex or the potent immunostimulatorlipopolysaccharide (LPS) at 50 micromolar concentrations (positivecontrol). Medium from assay wells was removed and centrifuged at 1300rpm in a microfuge for 5 minutes to pellet unattached dendritic cells.The supernatants were removed and treated with 10 microliters per ml ofCalbiochem (La Jolla, Calif.) protease inhibitor cocktail set I (Cat.No. 539131) and stored frozen prior to analysis. Samples were analyzedfor chemokine and cytokine expression by Searchlight Protein ArrayMultiplex ELISA assay [Pierce Biotechnology (Woburn, Mass.)].

Production of selected chemokines known to be essential in the cellularimmune response, CCL3, CCL4, CCL5, and CCL19 was evaluated, andproduction of IL-2, 11-8 and IL-12 was evaluated (FIGS. 5-11, whichillustrate the ability of R-DOTAP/E7 and S-DOTAP/E7 to induce productionof CCL3, CCL4, CCL5, CCL-19, IL-2, IL-8 and 1L-12), The figuresillustrate that the DOTAP/E7 complex containing the individualenantiomers of DOTAP induce cytokine and chemokine production by humandendritic cells. Both enantiorners however activate the immune system todifferent extents with the R-enantiomer exhibiting higher potency.

6. Kinetics of TC-.1 HPV-Positive Tumor Growth in Mice Treated withDOTAP/E7 Compositions at Varying Doses of Racemic Mixtures of DOTAP.

In FIG. 12, mice were subcutaneously injected with TC-1 cells on day 0in order to induce the growth of HPV-positive tumors, The DOTAP/E7compositions were comprised of racemic mixtures of DOTAP. The micereceived DOTAP/E7 compositions containing 10 11.g E7 peptidesubcutaneously on the opposite side of the abdomen on. day 6. DOTAPlipid concentration in the complex varied from 3 to 600 nmole (3, 15,30, 75, 150, 300, and 600 nmole), Low dose of DOTAP (15 nmole) showedpartial tumor inhibition effect (P<0.05) compared to the untreatedcontrol on day 23, while DOTAP at 30, 150 or 300 nmole exhibited anenhanced efficacy (P<0.01). DOTAP at 75 nmole showed the mostsignificant tumor regression effect (P<0.001). Again, mice given a highdose of DOTAP (600 nmole) did not show anti-tumor activity, confirmingthat DOTAP liposomes at a high dose might have induced a negativeregulation to the immune response. In addition, DOTAP liposomes at the100 nmole dose, but without E7 peptide, did not show significantinhibition of tumor growth, indicating that the anti-tumor effect wasantigen specific. Further, liposomes of1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), an anionic lipid,administered at the 150 nmole dose with antigen failed to significantlyinhibit tumor growth.

7. Kinetics of TC-1 HPV-Positive Tumor Growth in Mice Treated withR-DOTAP/E7 and S-DOTAP Compositions at Varying Doses of R and S DOTAP.

In FIGS. 13 and 14, mice were subcutaneously injected with TC-1 cells onday 0 in order to induce the growth of HPV-positive tumors. The micereceived R and S-DOTAP/E7 compositions containing 20 E7 peptidesubcutaneously on the opposite side of the abdomen on day 6. R orS-DOTAP lipid concentrations in the complex varied from 3 to 600 nmole(3, 15, 30, 75, 100, 125, 150, 300, and 600 nmole). Unlike the racemicmixture of DOTAP (FIG. 12), S-DOTAP complexes did not exhibit theability to inhibit tumor growth and no tumor regression was observed(FIG. 13). A dose response effect was however observed, and S-DOTAPcloses of 600 nmole induced the slowest tumor growth (P<0.05) comparedto untreated control on day 23. Referring to FIG. 14 the anti-tumoreffect of complexes containing R-DOTAP and antigen were similar to theeffect observed in the race/tile mixture (FIG. 12). 75-150 nmole dosesof R-DOTAP showed partial tumor inhibition effect (P<0.001) compared tothe untreated control on day 23, while R-DOTAP at 300 nmole exhibitedthe most significant tumor regression efficacy (P<0.0001). Again, micegiven a high dose of R-DOTAP (600 nmole) did not show significantanti-tumor activity, confirming that R-DOTAP liposomes at a high dosemight have induced a negative regulation to the immune response. E7peptide alone, did not show any inhibition of tumor growth (not shown).FIG. 15 shows the lipid dose-response curves for the tumor regressionefficacy of the various cationic lipid/E7 antigen complexes DOTAP,S-DOTAP, and R-DOTAP at 20 μg of the antigen and DOTAP at 10 μg of theantigen.

8. Induction of T Cell Proliferation by S-DOTAP and R-DOT.APCompositions.

We have previously demonstrated that in U.S. Provisional Application No.60/983,799, assigned to the assignee of the present application, thatDOTAP/E7 interacts directly with human T lymphocytes, leading to clonalexpansion and T cell activation. Those studies examined the ability ofracemic mixtures of DOTAP to stimulate clonal expansion of T cells. Inthose studies, enriched human lymphocytes obtained from an I-ILA-A2^(t)healthy donor were directly stimulated by medium, DOTAP alone, peptidealone or DOTAP/hE7. The stimulation was repeated three times with a7-day interval. Three days after the third stimulation, lymphocytestreated with DOTAP or DOTAP/E7 showed extensive expansion of T cellcolonies in culture in contrast to no clonal expansion in mediumcontrol. The expanded T-cells also demonstrated significant CTLactivity.

In those studies, the DOTAP-mediated T cell activation was furtherconfirmed by ERK phosphorylation in T cells. DOTAP-induced expression ofthe costimulatory molecule, CD86 on human T lymphocytes was alsoobserved. Those results suggested that DOTAP has a direct impact on Tcell activation via a MAP kinase mediated cell proliferation.

In the present studies, the induction of human T-cell proliferation bythe R and S enantiomers of DOTAP was investigated and confirmed usingpurified T-cells obtained from Lonza, Mass. R-DOTAP induced more T-cellproliferation than S-DOTAP and was similar in activity to the DOTAPracemic mixture.

Discussion

As described in U.S. Pat. No. 7,303,881, a broad class of cationiclipids can act as potent immunostimulators together with an antigen togenerate antigen specific immune responses in the treatment of disease.For example, U.S. Pat. No. 7,303,881 discloses that liposomes comprisedof cationic lipids activate dendritic cells as demonstrated by thestimulation by cationic lipids of the expression of costimulatorymolecules CD80/CD86 on DC2A dendritic cells (FIGS. 14A and 14B). Asshown in FIG. 14A of U.S. Pat. No. 7,303,881, the ability to stimulatethe expression of CD80/CD86 on DC2.4 cells by different cationicliposomes varies greatly. Lipofectamine®, a 3:1 (w/w) liposomeformulation of the polycationic lipid2,3-dioleyloxy-N-[2(spen-ninecarboxamido)ethyll-N,N-dimethyl-1-propanarniniumtrifluoroacetate (DOSPA) and the neutral lipid dioleoylphosphatidylethanolamine (DOPE), and liposomes prepared from0,0¹-dirnyristyl-N-lysyl aspartate (DMKE) and0,0′-dimyristyl-N-lysyl-glutamate (DMKD), strongly stimulated theexpression of CD80/CD86 by CD2.4 cells.

As further disclosed in U.S. Pat. No. 7,303,881, the ability ofdifferent cationic lipids to stimulate the expression of CD 80 on DC 2.4cells varied. Both hydrophilic head and the lipophilic tail of thelipids have significant effect on this ability. For example, the DXEPClipids with the ethyl phosphocholine (EPC) head groups appear, ingeneral, to be more potent than the DXTAP lipids with trimethylammoniumpropane (TAP) head group. Within the lipids bearing one particular headgroup structure, lipids with shorter(1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DXEPC-12:0),1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC-14:0)) orunsaturated (1,2-dioleoyl-sn-glycero-3-ethylphosphocholinc (DOEPC-18:1))acyl chains appear to be more potent than those with longer(1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DXEPC-16:0)) orsaturated (1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC-16:0))acyl chains, These data however, demonstrated that multiple cationiclipids were capable of stimulating the activation of dendritic cells.Studies reported in U.S. application Ser. No. 12/049,957 highlight themechanism by which cationic lipids act as immunostimulators.

Data from the abovementioned studies have led to the observation thatthe cationic lipids are not only efficient targeting and deliveryvehicles for antigens to APC of the immune system, but also function aspotent adjuvants under low dose composition ranges to directly influenceimmune system function through activation of MAP kinase dependentsignaling pathways with resultant production of immune system regulatorymolecules including cytokines and chemokines. A clear dose-responseeffect of cationic lipid on the immunostimulatory capabilities of theformulations have been demonstrated. It was demonstrated that uponreceiving the lipid/antigen complex, the particles were mainly taken upby dendritic cells, the major professional antigen presenting cells. Theinitiation of dendritic cell activation and migration to the draininglymph node facilitates immune responses against antigen specific TC-1tumors as demonstrated. Functional CD8⁺ T lymphocytes were generated inmice upon receiving a DOTAP/E7 injection and tumor sizes decreased andexhibited enhanced apoptosis, owing to the increasing number ofinfiltrating T cells in the tumor microenvironment. The resultingbell-shaped (activity decreases above and below the optimal dose)cationic lipid dose response curve demonstrated activity at very lowdoses, indicating that the activity of the cationic lipids as adjuvantsor immunostimulators is so potent that the EC₅₀ is as low as about 15nmole per injection. High doses of cationic lipids eliminate theimmunostimulatory activity. We have also demonstrated that when anantigen such as, for example, ovalbumin, is incorporated into thecationic liposomes and administered in a single subcutaneous injection,effective antibodies against the antigen are produced. Cationicliposomes can also induce expression of the co-stimulatory moleculesCD80 and CD83 and activate human dendritic cells. It is clear that atoptimal dose compositions, the cationic lipids and cationiclipid/antigen complexes in addition to effective delivery to thedendritic cells are potent activators of the immune system and providesimple, safe, and very efficient immunotherapies useful in preventingand treating diseases.

Based on an understanding of the mechanism of immunostimulation, furtherstudies were performed to evaluate the effect of chirality in cationiclipids and the immunostimulatory capability of cationic lipids, To thiseffect pure synthesized R and S enantiomers of DOTAP were utilized andcompared with the commonly utilized racemic mixture. Both R and Senantiomers of DOTAP were demonstrated to possess similar ability to theracemic DOTAP with regards to activation and maturation of dendriticcells. All three lipids induced dendritic cells to express theco-stimulatory molecules CD 80 CD 83 and CD 86.

An important characteristic of an immunostimulator capable of inducingcellular immune responses to disease is its ability to induce theproduction of critical chemokines and cytokines. As reported in theExample, significant differences were observed between the R and Senantiomers of DOTAP in their ability to induce chetnokine and cytokineproduction. R-DOTAP was observed to be a more potent immune activatorthan S-DOTAP. In all cases the potency of R-DOTAP was equivalent to orhigher than that of the DOATP racemic mixture.

In order to determine if the in-vitro potency in cytokine inductionwould translate to in-vivo therapeutic efficacy, three formulations,R-DOTAP/E7, S-DOTAP/E7 and DOTAP/E7 (racemic mixture) were evaluated fortheir ability to eradicate HPV-E7 positive tumors in tumor-bearing mice.Each formulation was evaluated at multiple lipid doses. As demonstratedin FIGS. 12-15, both R-DOTAP and DOTAP containing formulations exhibiteda bell-shaped lipid-dose response with strong E7 specific activityleading to tumor regression within specific optimal dose ranges. S-DOTAPcontaining formulations did not induce tumor regression under anycondition observed, although high lipid formulations slowed tumorgrowth.

It is therefore evident that the R enantiomer of DOTAP is responsiblefor the majority of the observed adjuvant effect of DOTAP. However, bothenantiomers are potent activators of dendritic cells leading tomaturation.

The studies reported above identify specific unique compositions andapplications of cationic lipids consisting of chiral lipid or mixturesof chiral lipids, which can be exploited to develop simple, costeffective, and much needed immunotherapies for several debilitatingdiseases.

As various changes could be made in the above-described aspects andexemplary embodiments without departing from the scope of the invention,it is intended that all matter contained in the above description shallbe interpreted as illustrative and not in a limiting sense.

To that end, while the examples primarily discuss enantiomers of thecationic lipid DOTAP, those skilled in the art will recognize that thiscationic lipids is merely exemplary and that the methods and mechanismsare applicable to other cationic lipids.

1. A method of enhancing an immunostimulatory effect in an immune systemof a mammal to respond prophylactically or therapeutically to aninfection, said method comprising the step of administering aimmunogenic composition to the mammal, wherein the composition comprisesa cationic lipid and an antigen, wherein the antigen consists of T-cellreceptor gamma alternate reading frame protein or (TARP) or Mucin 1 (MUC1).
 2. The method of claim 1, wherein the cationic lipid comprises achiral cationic lipid.
 3. The method of claim 2, wherein the chiralcationic lipid comprises 1,2-dioleoyl-3-trimethylammonium propane(DOTAP), N-1-(2,3-dioleoyloxy) propyl-N,N,N-trimethyl ammonium chloride(DOTMA), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), andcombinations thereof.
 4. The method of claim 3, wherein the compositionfurther comprises one or more therapeutic ingredients selected from thegroup consisting of drug molecules, cytokines, chemokines, polymercomplexes and pharmaceutical carriers.
 5. The method of claim 1, whereinthe antigen consists of TARP and antigenic fragments thereof.
 6. Themethod of claim 5, wherein the composition further comprises a nucleicacid molecule that encodes the amino acid sequence of TARP and antigenicfragments thereof.
 7. The method of claim 6 wherein the modified toincrease its hydrophobicity or wherein the protein domain of the antigenis modified to facilitate purification by adding a C-terminal proteintag that includes a poly-histidine tag and a proteolytic cleavagesequence for removal of the tags.
 8. The method of claim 1, wherein theantigens consists of MUC 1 and antigenic fragments thereof.
 9. Themethod of claim 8, wherein the composition further comprises a nucleicacid molecule that encodes an amino acid sequence of MUC 1 and antigenicfragments thereof.
 10. The method of claim 9, wherein the antigen ismodified to increase its hydrophobicity or wherein the protein domain ofthe antigen is modified to facilitate purification by adding aC-terminal protein tag that includes a poly-histidine tag and aproteolytic cleavage sequence for removal of the tags.
 11. The method ofclaim 1, wherein the cationic lipid comprises DOTAP.
 12. The method ofclaim 11, wherein the cationic lipid consists of R-DOTAP.
 13. The methodof claim 1, wherein the mammal is a human.
 14. The method of claim 1,wherein the wherein the composition is administered as an aerosol or asa liquid solution for intratumoral, intraarterial, intravenous,intratracheal, intraperitoneal, subcutaneous, or intramuscularadministration.
 15. The method of claim 1, wherein the immunostimulatoryeffect includes production of immune system regulatory molecules.
 16. Apharmaceutical composition comprising an immunologically activeenantiomer of a cationic lipid component, wherein the cationic lipidcomponent consists of R-DOTAP, and an antigen, wherein the antigenconsists of T-cell receptor gamma alternate reading frame protein or(TARP) or Mucin 1 (MUC 1), and wherein the composition is effective toactivate an immune system in a patient.
 17. The pharmaceuticalcomposition of claim 16, further comprising additional lipids selectedfrom the group consisting of lyso lipids, lysophosphatidylcholine,1-oleoyl lysophosphatidylcholine, cholesterol, neutral phospholipids,dioleoyl phosphatidyl ethanolamine (DOPE), dioleoyl phosphatidylcholine(DOPC), lipophilic surfactants, Tween-80 and PEG-PE, and combinationsthereof.
 18. The pharmaceutical composition of claim 16, wherein theantigen consists of TARP, or antigenic fragments thereof.
 19. Thepharmaceutical composition of claim 18, wherein the composition furthercomprises a nucleic acid molecule that encodes an amino acid sequence ofTARP, or antigenic fragments thereof.
 20. The pharmaceutical compositionof claim 16, wherein the antigen consists of MUC 1, or antigenicfragments thereof.
 21. The pharmaceutical composition of claim 20,wherein the composition further comprises a nucleic acid molecule thatencodes an amino acid sequence of MUC 1, or antigenic fragments thereof.22. The pharmaceutical composition of claim 16, wherein the antigenincludes an antigen modified to increase its hydrophobicity.